The pathogenicity of Corynebacterium diphteriae, causing diptheria in man, is primarily due to the production of potent proteinaceous exotoxin which inhibits cellular protein synthesis in susceptible animals and man. The genetic information for synthesis of this toxin resides in the DNA of certain corynebacteriophages. This system is an excellent model of how virus-infected pathogenic bacteria may direct the synthesis of microbial toxins. Cellular factors in the growth medium and the host bacterial strain determine how much toxin is synthesized. The singular most important factor is presence of iron in the medium which depresses toxin synthesis. The objective of the proposed research is to determine (1) quantity of toxin mRNA present in cells cultured in absence and presence of iron, and in absence of apparent growth when toxin accumulation in the medium is maximal, (2) the presence of restriction endonuclease fragment(s) of toxigenic phage Bv tox+ which hybridizes to mRNA from toxigenic cells, and (3) the position of tox gene on the restriction endonuclease-derived physical map of Bv tox+ phage. A cell-free-protein synthesizing system from E. coli will be used to compare toxin synthesis by mRNAs extracted from cells cultured in high and low iron concentration. 3H-RNA derived from the above bacterial cultures will be hybridized to toxigenic to excreate diptheriae toxin. The physical map of Bv tox+ phage will be determined using restriction endonucleases. The cellular 3H-RNA hybridizing to specific restriction enonuclease DNA fragments will be identified.